Hemagglutinin Modifications for Improved Influenza Vaccine Production

ABSTRACT

Mutation of adenosine methylation sites in nucleic acids encoding influenza virus hemagglutinin are provided that result in increases in vRNA, mRNA, and protein expression over time and increases in infectious titers when produced in mammalian cells.

INTRODUCTION

This patent application claims the benefit of priority from U.S.Provisional Ser. No. 63/073,614, filed Sep. 2, 2020, the content ofwhich is incorporated herein by reference in its entirety.

This invention was made with government support under grant no. AI106700awarded by the National Institutes of Health. The government has certainrights in this invention.

BACKGROUND

The covalent modification of individual bases on mRNA transcripts hasemerged as a potentially critical mechanism for the post-transcriptionalregulation of gene expression. Analysis of the cellularepitranscriptome, defined as internal single nucleotide modificationsthat do not alter the mRNA sequence, has identified at least tendifferent modifications, of which the most prevalent is the addition ofa methyl group to the N⁶position of adenosine, referred to as m⁶A. m⁶Ais added co-transcriptionally to nuclear pre-mRNAs by a protein complexcomposed minimally of the methyltransferase METTL3 and two co-factors,METTL14 and WTAP, known collectively as m⁶A “writers.” Once mRNAs haveentered the cytoplasm, they encounter three m⁶A “reader” proteins calledYTHDF1, YTHDF2 and YTHDF3, which are thought to mediate many of thephenotypic effects exerted by m⁶A.

In addition to the important role played by m⁶A in regulating cellularmRNA function, m⁶A has also been detected on every viral mRNA transcriptexamined so far including mRNAs encoded by several cytoplasmic RNAviruses. The first virus found to express mRNAs bearing internal m⁶Aresidues was influenza A virus, wherein eight m⁶As were detected bybiochemical analysis of the HA mRNA segment (Krug, et al. (1976) J.Virol. 20(1):45-53; Narayan, et al. (1987) Mol. Cell. Biol.7(4):1572-5). However, these m⁶A residues were not mapped and noexamination of how m⁶A affects influenza gene expression or replicationhas been reported.

Global inhibition of m⁶A addition by depleting intracellular levels ofthe methyl donor S-adenosylmethionine with 3-deazaadenosine (DAA) hasbeen shown to inhibit influenza A virus (Bader, et al. (1978) Virology89(2):494-505; Fustin, et al. (2013) Cell 155(4):793-806). In addition,mutant forms of influenza A virus in which eight prominent m⁶A sites onthe HA mRNA/cRNA plus strand, or nine m⁶A sites on the HA vRNA minusstrand, have been prepared and shown to express lower levels of HA mRNAand be significantly less pathogenic when introduced into mice(Courtney, et al. (2017) Cell Host Microbe 22(3):377-386.e5).

SUMMARY OF THE INVENTION

This invention provides a modified nucleic acid molecule encodinginfluenza virus hemagglutinin, wherein said nucleic acid moleculeincludes (a) a C->T mutation in the third position of codon 433 of thehemagglutinin; (b) an A->T mutation in the first position of codon 436of the hemagglutinin; or (c) a combination of (a) and (b). In someembodiments, the modified nucleic acid molecule encodes a modifiedhemagglutinin protein having a Thr436Ser amino acid substitution. Avector and host cell harboring the modified nucleic acid molecule arealso provided as is a modified influenza virus hemagglutinin proteinhaving a Thr436Ser amino acid substitution. Vaccines composed of themodified nucleic acid molecule or modified influenza virus hemagglutininprotein are also embraced by this invention as is a method of theproduction of influenza virus in a mammalian cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows vRNA and mRNA HA expression levels after a singlereplication cycle of wild-type and mutant 2 virus as determined byRT-PCR.

FIG. 2 shows infectious viral titers (Pfu) after a single replicationcycle of wild-type and mutant 2 virus. ** P<0.01 vs PR8 wild-type,one-way ANOVA.

FIG. 3 shows relative vRNA and mRNA HA expression levels after multiplereplication cycles of wild-type and mutant 2 virus as determined byRT-PCR.

FIG. 4 shows infectious viral titers (Pfu) after multiple replicationcycles of wild-type and mutant 2 virus.

FIG. 5 shows viral lung titers (Panel A) and percent active infectionarea (Panel B) in mice infected with wild-type and mutant 2 virus atdays 2, 4 and 6 post-infection.

FIG. 6 shows cytokine and chemokine levels in mice infected withwild-type and mutant 2 virus.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that mutation of specific sites associated withadenosine methylation (m⁶A) results in increases in vRNA, mRNA, andprotein expression over time compared to wild-type levels in a humancell line. These increases occur within a single replication cycle andare maintained over multiple replication rounds. Increases in viral RNAand protein expression also result in a significant increase ininfectious titers, immunological responses and lethality in a mousemodel. Advantageously, increasing yield in human cell lines can reducethe cost of vaccine production in mammalian cells. Accordingly, thisinvention provides novel influenza HA nucleic acid molecules andproteins with selected mutations at m⁶A, which are of use in theproduction of influenza virus vaccines, in particular in mammaliancells.

Influenza viruses are members of the orthomyxoviridae family and areclassified into three major distinct types (A, B, and C), based onantigenic differences between their nucleoprotein (NP) and matrix (M)protein. Influenza virions are composed of an internal ribonucleoproteincore (a helical nucleocapsid) containing a single-stranded, segmentedRNA genome, and an outer lipoprotein envelope lined inside by a matrixprotein (M1). The segmented genome of influenza A virus includes eightmolecules of linear, negative polarity, single-stranded RNAs that encodethe RNA-dependent RNA polymerase proteins (PB2, PB1 and PA) andnucleoprotein (NP), which form the nucleocapsid; the matrix and ionchannel proteins (M1, M2); two surface glycoproteins (hemagglutinin (HA)and neuraminidase (NA)); and nonstructural proteins (NS1 and NS2) inaddition to other accessory proteins. Transcription and replication ofthe genome takes place in the nucleus and assembly takes place at theplasma membrane.

Hemagglutinin. HA is a viral surface glycoprotein comprisingapproximately 560 amino acids and representing 25% of the total virusprotein. HA is responsible for adhesion of the viral particle to, andits penetration into, a host cell in the early stages of infection.There are 18 known HA subtypes, categorized as an H1, H2, H3, H4, H5,H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 or H18 subtype.An exemplary wild-type influenza A virus HA protein has the amino acidsequence:

(SEQ ID NO: 1) MKANLLVLLSALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNIYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNTPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQILAIYSTVASSLVLLVSLGAIS FWMCSNGSLQCRICI.

In some embodiments, the influenza A virus HA protein shares at least75% (e.g., any number between 75% and 100%, inclusive of, e.g., 70%,80%, 85%, 90%, 95%, 99%, and 100%) identity to an amino acid sequence ofSEQ ID NO:1. Representative nucleic acid sequences encoding wild-typeinfluenza A virus HA proteins are presented in Table 1.

TABLE 1 Strain Accession SEQ ID No. Strain Number NO: 1A/California/04/2009 FJ966082 2 2 A/California/07/2009 FJ969540 3 3A/Canada-AB/RV1644/2009 GQ465679 4 4 A/California/06/2009 FJ966960 5 5A/Omsk/02/2009 GU211235 6 6 A/Denmark/523/2009 CY043334 7 7A/Shanghai/1/2009 GQ225357 8 8 A/New York/3324/2009 CY043195 9 9A/Shanghai/143T/2009 GQ411907 10 10 A/Wisconsin/629-D00008/2009 CY05104711 11 A/Beijing/3/2009 GQ225381 12 12 A/Osaka/1/2009 GQ219578 13 13A/Korea/01/2009 GQ131023 14 14 A/England/195/2009 GQ166661 15 15A/Hamburg/4/2009 GQ166213 16 16 A/New York/3177/2009 CY041597 17 17A/Kansas/03/2009 GQ168644 18 18 A/Moscow/WRAIR4316N/2011 CY098052 19 19A/Netherlands/602/2009 CY039527 20 20 A/Santo Domingo/0573N/2009CY041983 21 21 A/Brawley/40081/2009 CY043086 22 22 A/Vladivostok/01/2009GU211219 23 23 A/Craven/WR0019/2009 CY049820 24 24 A/Nanjing/2/2009GQ455032 25 25 A/Nebraska/02/2009 GQ377082 26 26A/Wisconsin/629-D00022/2009 CY051223 27 27 A/Colorado/03/2009 GQ11711928 28 A/Sichuan/1/2009 GQ166223 29 29 A/Minnesota/02/2009 GQ338364 30 30A/Indiana/09/2009 GQ117097 31 31 A/Amagasaki/1/2009 GQ219574 32 32A/Sakai/1/2009 GQ267839 33 33 A/Himeji/1/2009 GQ261272 34 34A/Kobe/1/2009 GQ219577 35 35 A/Beijing/501/2009 GQ223408 36 36A/Utsunomiya/1/2009 GQ334355 37 37 A/Hunan/SWL3/2009 GQ463200 38 38A/Netherlands/2631_1202/2010 JF906183 39 39 A/Ohio/07/2009 GQ117100 4040 A/Shanghai/3162T/2011 JN631050 41 41 A/Volgograd/CRIE-DMV/2011JN714508 42 42 A/Finland/65/2011 JN601109 43 43 A/Assam/2220/2009JN600356 44 44 A/Assam/2590/2010 JN600357 45 45 A/Cambodia/U127/2010JN588791 46 46 A/Thailand/CU-B5/2009 GQ866951 47 47 A/Taiwan/T1773/2009CY044220 48 48 A/Silver Spring/SP509/2009 CY044179 49 49A/Nanjing/3/2009 GU198201 50 50 A/Shizuoka/759/2009 GQ334346 51 51A/Shiga/3/2009 GQ287623 52 52 A/San Salvador/0169T/2009 CY049891 53 53A/Cherry Point/WR0100/2009 CY049859 54 54 A/Taiwan/1018/2011 JN187143 5555 A/Boston/DOA14/2011 CY111206 56 56 A/Thailand/CU-H2911/2011 CY08946357 57 A/Mexico/InDRE3740/2011 CY116642 58 58 A/California/NHRC0001/2011CY092880 59 59 A/Brazil/AVS08/2011 CY120747 60 60 A/SouthCarolina/09/2009 GQ117056 61 61 A/Pune/NIV6447/2009 GU292353 62 62A/India/Blore/2010 JF293316 63 63 A/India/GWL_DSC/2010 JF293315 64 64A/India/GWL01/2011 JQ319658 65 65 A/India/GWL02/2011 JQ319657 66 66A/Delhi/NIV3704/2009 GU292349 67 67 A/Pune/NIV9355/2009 GU292355 68 68A/Blore/NIV236/2009 GU292346 69 69 A/Pune/NIV10278/2009 GU292344 70 70A/Mum/NIV9945/2009 GU292356 71 71 A/Delhi/NIV3610/2009 GU292348 72 72A/Mum/NIV5442/2009 GU292351 73 73 A/Blore/NIV310/2009 GU292347 74 74A/Pune/NIV8489/2009 GU292354 75 75 A/Pune/NIV6196/2009 GU292352 76 76A/Pune/NIV10604/2009 GU292345 77 77 A/Hyd/NIV51/2009 GU292350 78

In some aspects of this invention, the nucleic acid molecule encodingthe HA protein lacks one or more N6-methyl-adenosine (m⁶A) nucleotides.In particular aspects, the nucleic acid molecule encoding the HA proteinhas been modified to include a C->T mutation in the third position ofthe codon at position 433 of the HA protein amino acid sequence (i.e.,mutation of the codon GAC to GAT); an A->T mutation in the firstposition of codon Thr436 of the HA protein amino acid sequence; or acombination thereof, thereby eliminating the m⁶A and providing asignificant increase in HA RNA expression in mammalian cells. In otheraspects, the nucleic acid molecule encoding the HA protein has beenmodified to include a C->T mutation in the third position of the codonat position 433 of the HA protein amino acid sequence of SEQ ID NO:1; anA->T mutation in the first position of codon Thr436 of the HA proteinamino acid sequence of SEQ ID NO:1; or a combination thereof. Withreference to SEQ ID NO:l, other aspects of this invention include anucleic acid molecule encoding a modified HA protein that has an aminoacid substitution at amino acid residue 436. In some aspects, thisinvention provides a nucleic acid molecule encoding a modified HAprotein that has an amino acid substitution at amino acid residue 436 ofSEQ ID NO:1. In certain aspects, the nucleic acid molecule of theinvention encodes a modified HA protein having a Thr436Ser amino acidsubstitution.

For the purposes of this invention, a “nucleic acid molecule” refers toa single-stranded or double-stranded deoxyribonucleotide orribonucleotide polymer, chimera or analogue thereof, or a characterstring representing such, depending on context. The term “nucleic acidmolecule” encompasses any physical string of monomer units that can becorresponded to a string of nucleotides, including a polymer ofnucleotides (e.g., a typical DNA or RNA polymer), PNAs, modifiedoligonucleotides (e.g., oligonucleotides comprising bases that are nottypical to biological RNA or DNA in solution, such as 2′-O-methylatedoligonucleotides), and the like. A nucleic acid can be e.g.,single-stranded or double-stranded. A nucleic acid molecule is deemed tobe “modified” when the sequence has been altered by one or morenucleotides with respect to a reference sequence. Similarly, a“modified” or “variant” protein has an amino acid sequence that has beenaltered by one or more amino acids with respect to a reference sequence(e.g., a wild-type protein).

Cleavage of the virus HA0 precursor into the HA1 and HA2 subfragments isa necessary step for the virus to infect a cell. Thus, cleavage isrequired to convert new virus particles in the host cells into virionscapable of infecting new cells. Cleavage is known to occur duringtransport of the integral HA0 membrane protein within the infected cellas well as extracellularly. In the course of transport, hemagglutininundergoes a series of co- and post-translational modifications which caninclude proteolytic cleavage of the precursor HA into the amino-terminalfragment HA1 and the carboxy terminal HA2. One of the primarydifficulties in growing influenza strains in primary tissue culture orestablished cell lines arises from the requirement for proteolyticcleavage activation of the influenza hemagglutinin in the host cell.

Although it is known that an uncleaved HA can mediate attachment of thevirus to its sialic acid-containing receptors on a cell surface, it isnot capable of the next step in the infectious cycle, which is fusion.It has been reported that exposure of the hydrophobic amino terminus ofthe HA2 by cleavage is required so that it can be inserted into thetarget cell, thereby forming a bridge between virus and target cellmembrane. This process is followed by fusion of the two membranes andrelease of the viral genome into the target cell.

Proteolytic activation of HA involves cleavage at an arginine residue bya trypsin-like endoprotease, which is often an intracellular enzyme thatis calcium dependent and has a neutral pH optimum. Since the activatingproteases are cellular enzymes, the infected cell type determineswhether the HA is cleaved. The HA of the mammalian influenza viruses andthe nonpathogenic avian influenza viruses are susceptible to proteolyticcleavage only in a restricted number of cell types. On the other hand,HA of pathogenic avian viruses among the H5 and H7 subtypes are cleavedby proteases present in a broad range of different host cells. Thus,there are differences in host range resulting from differences inhemagglutinin cleavability which are correlated with the pathogenicproperties of the virus.

The differences in cleavability are due to differences in the amino acidsequence of the cleavage site of the HA. Sequence analyses show that theHA1 and HA2 fragments of the HA molecule of the non-pathogenic avian andall typical mammalian influenza viruses are linked by a single arginine.In contrast, the pathogenic avian strains have a sequence of severalbasic amino acids at the cleavage site with the common denominator beinglysine-arginine or arginine-arginine, e.g., RRRK. The hemagglutinins ofall influenza viruses are cleaved by the same general mechanismresulting in the elimination of the basic amino acids.

Neuraminidase. NA is a second membrane glycoprotein of the influenza Aviruses. The presence of viral NA has been shown to be important forgenerating a multi-faceted protective immune response against aninfecting virus. NA is a 413 amino acid protein encoded by a gene of1413 nucleotides. Eleven different NA subtypes have been identified ininfluenza viruses (N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 and N11). NAis involved in the destruction of the cellular receptor for the viral HAby cleaving terminal sialic acid carbohydrate moieties on the surfacesof infected cells. NA also cleaves sialic acid residues from viralproteins, preventing aggregation of viruses. Using this mechanism, it ishypothesized that NA facilitates release of viral progeny by preventingnewly formed viral particles from accumulating along the cell membrane,as well as by promoting transportation of the virus through the mucuspresent on the mucosal surface. NA is an important antigenic determinantthat is subject to antigenic variation. Representative strains andnucleic acid sequences encoding wild-type influenza A virus NA proteinsare presented in and Table 2.

TABLE 2 Strain No. Strain Accession No. 1 A/California/04/2009 FJ9660842 A/Pune/NIV6447/2009 GU292385 3 A/India/Blore/2010 JF265672 4A/India/GWL_DSC/2010 JF265671 5 A/India/GWL01/2011 JX262201 6A/India/GWL02/2011 JX262202 7 A/California/07/2009 GQ377078 8A/Canada-AB/RV1644/2009 GQ465702 9 A/California/06/2009 FJ971075 10A/Blore/NIV236/2009 GU292381 11 A/Blore/NIV310/2009 GU292382 12 A/NewYork/3324/2009 CY043197 13 A/Pune/NIV8489/2009 GU292386 14A/Shanghai/143T/2009 GQ411905 15 A/Pune/NIV6196/2009 GU292384 16A/Wisconsin/629-D00008/2009 CY051049 17 A/Beijing/3/2009 GQ225383 18A/Hyd/NIV51/2009 GU292383 19 A/Osaka/1/2009 GQ220734 20 A/Korea/01/2009GQ132185 21 A/England/195/2009 GQ166659 22 A/New York/3177/2009 CY04159923 A/Moscow/WRAIR4316N/2011 CY098054 24 A/Netherlands/602/2009 CY03952825 A/Santo Domingo/0573N/2009 CY041985 26 A/Brawley/40081/2009 CY04308827 A/Vladivostok/01/2009 GU211221 28 A/Craven/WR0019/2009 CY049822 29A/Nanjing/2/2009 GQ455034 30 A/Nebraska/02/2009 GQ221802 31A/Wisconsin/629-D00022/2009 CY051225 32 A/Colorado/03/2009 GQ221813 33A/Sichuan/1/2009 GQ166224 34 A/Minnesota/02/2009 GQ117071 35A/Indiana/09/2009 GQ117094 36 A/Amagasaki/1/2009 GQ220730 37A/Sakai/1/2009 GQ261274 38 A/Himeji/1/2009 GQ261273 39 A/Kobe/1/2009GQ220733 40 A/Beijing/501/2009 GQ223415 41 A/New York/4735/2009 CY05166542 A/Canada-PQ/RV1758/2009 GQ465703 43 A/Utsunomiya/1/2009 GQ334357 44A/Hunan/SWL3/2009 GQ463202 45 A/Thailand/CU-B5/2009 GQ866953 46A/Taiwan/T1773/2009 CY044222 47 A/Silver Spring/SP509/2009 CY044181 48A/Nanjing/3/2009 GU198203 49 A/Shizuoka/759/2009 GQ334348 50A/Shiga/3/2009 GQ287624 51 A/Mexico City/WR1100N/2009 CY049997 52 A/SanSalvador/0169T/2009 CY049893 53 A/New Bern/WR0670/2009 CY049925 54A/Cherry Point/WR0100/2009 CY049861 55 A/Taiwan/1018/2011 JN187201 56A/Boston/DOA14/2011 CY111208 57 A/Sydney/DD3-58/2011 CY092858 58A/Thailand/CU-H2911/2011 CY089465 59 A/Mexico/InDRE3740/2011 CY115448 60A/Missouri/NHRC0001/2011 CY092419 61 A/Cheboksary/IIV-92/2011 JN70479362 A/Tomsk/IIV-19/2012 JQ768355 63 A/California/NHRC0001/2011 CY09288264 A/Brazil/AVS08/2011 CY120749 65 A/South Carolina/09/2009 GQ221795

Internal Genes of Influenza. In addition to the surface proteins HA andNA, influenza virus includes six additional internal genes, which giverise to eight or more different proteins, including but not limited topolymerase genes PB1, PB2 and PA, matrix proteins M1 and M2,nucleoprotein (NP), and non-structural (NS) proteins NS1 and NS2.

In order to be packaged into progeny virions, viral RNA is transportedfrom the nucleus as a ribonucleoprotein complex composed of the threeinfluenza virus polymerase proteins, the nucleoprotein (NP), and theviral RNA, in association with the influenza virus matrix 1 (M1) proteinand nuclear export protein. The M1 protein that lies within the envelopefunctions in assembly and budding.

A limited number of M2 proteins are integrated into the virions. Theyform tetramers having H+ ion channel activity, and, when activated bythe low pH in endosomes, acidify the inside of the virion, facilitatingits uncoating.

NS1 protein, a nonstructural protein, has multiple functions, includingregulation of splicing and nuclear export of cellular mRNAs as well asstimulation of translation. The major function of NS1 seems to be tocounteract the interferon activity of the host, since an NS1 knockoutvirus was viable although it grew less efficiently than the parent virusin interferon-nondefective cells.

NS2 protein has been detected in virus particles. The average number ofNS2 proteins in a virus particle was estimated to be 130-200 molecules.An in vitro binding assay shows direct protein-protein contact betweenM1 and N52. NS2-M1 complexes were also detected by immunoprecipitationin virus-infected cell lysates. The NS2 protein, known to exist invirions, plays a role in the export of RNP from the nucleus throughinteraction with M1 protein.

Representative strains and nucleic acid sequences encoding wild-typeinfluenza A virus PB1, PB2, and PA proteins are presented in Table 3 andrepresentative strains and nucleic acid sequences encoding wild-typeinfluenza A virus matrix protein (MP), NP, and NS proteins are presentedin and Table 4.

TABLE 3 Strain PB2 PB1 PA A/California/04/2009 FJ966079 FJ966080FJ966081 A/Pune/NIV6447/2009 GU292361 GU292367 GU292373A/India/Blore/2010 JF265677 JF764086 JF265676 A/India/GWL_DSC/2010JF265678 JF764085 JF265675 A/India/GWL01/2011 JX262203 JX262205 JX262207A/India/GWL02/2011 JX262204 JX262206 JX262208 A/California/07/2009FJ969530 FJ969531 FJ969529 A/Canada-AB/RV1644/2009 GQ465667 GQ465751GQ465739 A/California/06/2009 FJ966963 FJ966965 FJ966964A/Blore/NIV236/2009 GU292357 GU292363 GU292369 A/Blore/NIV310/2009GU292364 GU292358 GU292370 A/NewYork/3324/2009 CY043202 CY043201CY043200 A/Pune/NIV8489/2009 GU292362 GU292368 GU292374A/Shanghai/143T/2009 GQ340061 GQ340062 GQ411906 A/Pune/NIV6196/2009GU292360 GU292366 GU292372 A/Wisconsin/629-D00008/ CY051054 CY051053CY051052 2009 A/Beijing/3/2009 GQ225378 GQ225379 GQ225380A/Hyd/NIV51/2009 GU292359 GU292365 GU292371 A/Osaka/1/2009 GQ222055GQ222046 GQ222037 A/Korea/01/2009 GQ160811 GQ160813 GQ160812A/England/195/2009 GQ166656 GQ166655 GQ166654 A/NewYork/3177/2009CY041604 CY041603 CY041602 A/Moscow/WRAIR4316N/ CY098049 CY098050CY098051 2011 A/Netherlands/602/2009 CY046940 CY046941 CY046942A/SantoDomingo/0573N/2009 CY041980 CY041981 CY041982A/Brawley/40081/2009 CY043083 CY043084 CY043085 A/Vladivostok/01/2009GU211226 GU211225 GU211224 A/Craven/WR0019/2009 CY049817 CY049818CY049819 A/Nanjing/2/2009 GQ455029 GQ455030 GQ455031 A/Nebraska/02/2009GQ200260 GQ168875 GQ457496 A/Wisconsin/629-D00022/ CY051230 CY051229CY051228 2009 A/Colorado/03/2009 GQ200263 GQ168884 GQ200264A/Sichuan/1/2009 GQ166228 GQ166227 GQ166226 A/Minnesota/02/2009 GQ117070GQ117069 GQ457488 A/Indiana/09/2009 GQ168870 GQ117093 GQ117095A/Amagasaki/1/2009 GQ222050 GQ222041 GQ222032 A/Sakai/1/2009 GQ267845GQ267844 GQ267843 A/Himeji/1/2009 GQ267838 GQ267837 GQ267836A/Kobe/1/2009 GQ222054 GQ222045 GQ222036 A/Beijing/501/2009 GQ223412GQ223413 GQ223414 A/NewYork/4735/2009 CY051670 CY051669 CY051668A/Canada-PQ/RV1758/2009 GQ465668 GQ465752 GQ465740 A/Utsunomiya/1/2009GQ334354 GQ334361 GQ334360 A/Hunan/SWL3/2009 GQ463197 GQ463198 GQ463199A/Thailand/CU-B5/2009 GQ866948 GQ866949 GQ866950 A/Taiwan/T1773/2009CY044217 CY044218 CY044219 A/SilverSpring/SP509/2009 CY044176 CY044177CY044178 A/Nanjing/3/2009 GU198198 GU198199 GU198200 A/Shizuoka/759/2009GQ334352 GQ334353 GQ334351 A/Shiga/3/2009 GQ324571 GQ324570 GQ324569A/MexicoCity/WR1100N/ CY049992 CY049993 CY049994 2009A/SanSalvador/0169T/2009 CY049888 CY049889 CY049890A/NewBern/WR0670/2009 CY049920 CY049921 CY049922A/CherryPoint/WR0100/2009 CY049856 CY049857 CY049858 A/Taiwan/1018/2011JN187172 JN187230 JN187259 A/Boston/DOA14/2011 CY111213 CY111212CY111211 A/Sydney/DD3-58/2011 CY092863 CY092862 CY092861A/Thailand/CU-H2911/2011 CY089460 CY089461 CY089462A/Mexico/InDRE3740/2011 CY120019 CY120020 CY116630A/Missouri/NHRC0001/2011 CY092424 CY092423 CY092422A/Cheboksary/IIV-92/2011 JN703379 JN703380 JN704790 A/Tomsk/IIV-19/2012JQ768350 JQ768351 JQ768352 A/California/NHRC0001/2011 CY092887 CY092886CY092885 A/Brazil/AVS08/2011 CY120754 CY120753 CY120752A/SouthCarolina/09/2009 GQ200222 GQ168854 GQ457472

TABLE 4 Strain NP MP NS A/California/04/2009 FJ966083 FJ966085 FJ966086A/Pune/NIV6447/2009 GU292379 GU292391 GU292397 A/India/Blore/2010JF265674 JF764082 JF764084 A/India/GWL_DSC/2010 JF265673 JF510037JF764083 A/India/GWL01/2011 JX262209 JX262211 JX262213A/India/GWL02/2011 JX262210 JX262212 JX262214 A/California/07/2009FJ969536 FJ969537 FJ969528 A/Canada-AB/RV1644/2009 GQ465715 GQ465691GQ465727 A/California/06/2009 FJ966961 FJ966962 FJ971074A/Blore/NIV236/2009 GU292375 GU292387 GU292393 A/Blore/NIV310/2009GU292376 GU292388 GU292394 A/NewYork/3324/2009 CY043198 CY043196CY043199 A/Pune/NIV8489/2009 GU292380 GU292392 GU292398A/Shanghai/143T/2009 GQ411909 GQ340064 GQ340063 A/Pune/NIV6196/2009GU292378 GU292390 GU292396 A/Wisconsin/629-D00008/ CY051050 CY051048CY051051 2009 A/Beijing/3/2009 GQ225382 GQ225384 GQ225385A/Hyd/NIV51/2009 GU292377 GU292389 GU292395 A/Osaka/1/2009 GQ223421GQ222028 GQ223430 A/Korea/01/2009 GQ131024 GQ131025 GQ131026A/England/195/2009 GQ166658 GQ166660 GQ166657 A/NewYork/3177/2009CY041600 CY041598 CY041601 A/Moscow/WRAIR4316N/ CY098053 CY098055CY098056 2011 A/Netherlands/602/2009 CY046943 CY046944 CY046945A/SantoDomingo/0573N/2009 CY041984 CY041986 CY041987A/Brawley/40081/2009 CY043087 CY043089 CY043090 A/Vladivostok/01/2009GU211222 GU211220 GU211223 A/Craven/WR0019/2009 CY049821 CY049823CY049824 A/Nanjing/2/2009 GQ455033 GQ455035 GQ455036 A/Nebraska/02/2009GQ117104 GQ457495 GQ117106 A/Wisconsin/629-D00022/ CY051226 CY051224CY051227 2009 A/Colorado/03/2009 GQ117117 GQ457502 GQ377090A/Sichuan/1/2009 GQ166225 GQ166229 GQ166230 A/Minnesota/02/2009 GQ117068GQ117073 GQ117072 A/Indiana/09/2009 GQ117092 GQ117096 GQ168871A/Amagasaki/1/2009 GQ223416 GQ222023 GQ223425 A/Sakai/1/2009 GQ267841GQ267840 GQ267842 A/Himeji/1/2009 GQ267834 GQ267833 GQ267835A/Kobe/1/2009 GQ223420 GQ222027 GQ223429 A/Beijing/501/2009 GQ223410GQ223409 GQ223411 A/NewYork/4735/2009 CY051666 CY051664 CY051667A/Canada-PQ/RV1758/2009 GQ465716 GQ465692 GQ465728 A/Utsunomiya/1/2009GQ334358 GQ334356 GQ334359 A/Hunan/SWL3/2009 GQ463201 GQ463203 GQ463204A/Thailand/CU-B5/2009 GQ866952 GQ866954 GQ866955 A/Taiwan/T1773/2009CY044221 CY044223 CY044224 A/SilverSpring/SP509/2009 CY044180 CY044182CY044183 A/Nanjing/3/2009 GU198202 GU198204 GU198205 A/Shizuoka/759/2009GQ334349 GQ334347 GQ334350 A/Shiga/3/2009 GQ324567 GQ324566 GQ324568A/MexicoCity/WR1100N/ CY049996 CY049998 CY049999 2009A/SanSalvador/0169T/2009 CY049892 CY049894 CY049895A/NewBern/WR0670/2009 CY049924 CY049926 CY049927A/CherryPoint/WR0100/2009 CY049860 CY049862 CY049863 A/Taiwan/1018/2011JN187288 JN187317 JN187346 A/Boston/DOA14/2011 CY111209 CY111207CY111210 A/Sydney/DD3-58/2011 CY092859 CY092857 CY092860A/Thailand/CU-H2911/2011 CY089464 CY089466 CY089467A/Mexico/InDRE3740/2011 CY115447 CY115449 CY115450A/Missouri/NHRC0001/2011 CY092420 CY092418 CY092421A/Cheboksary/IIV-92/2011 JN704792 JN704794 JN704795 A/Tomsk/IIV-19/2012JQ768354 JQ768356 JQ768357 A/California/NHRC0001/2011 CY092883 CY092881CY092884 A/Brazil/AVS08/2011 CY120750 CY120748 CY120751A/SouthCarolina/09/2009 GQ117052 GQ221796 GQ117054

Reverse Genetics and Reassortant Viruses. Techniques to isolate andmodify specific nucleic acids and proteins are well-known to those ofskill in the art. In accordance with the present invention there may beemployed conventional molecular biology, microbiology, and recombinantDNA techniques within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989; DNACloning. A Practical Approach, Volumes I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1985); Transcription andTranslation (B. D. Hames & S. J. Higgins, eds. 1984); Immobilized CellsAnd Enzymes (IRL Press, 1986); Ausubel, F. M. et al. (eds.). CurrentProtocols in Molecular Biology. John Wiley & Sons, Inc., 1994. Thesetechniques include site-directed mutagenesis employing oligonucleotideswith altered nucleotides for generating PCR products with mutations.

In one aspect, the present invention includes a method for theproduction of influenza virus, which includes the steps of (a)transfecting host cells with a modified nucleic acid molecule encodingan HA protein as described herein along with nucleic acid segments NA,PB1, PB2, PA, MP, NP, and NS; (b) incubating the host cells underconditions that allow for influenza virus to be produced; and (c)isolating the influenza virus from the host cells. The generalmethodology for the production of influenza viruses and vaccines thereofby reverse genetics methodologies as described herein and known in theart.

The mechanism of influenza viral RNA transcription is unique. The 5′ capfrom cellular mRNAs is cleaved by a viral endonuclease and used as aprimer for transcription by the viral transcriptase. Six of eight RNAsegments are transcribed into mRNAs in a monocistronic manner andtranslated into HA, NA, NP, PB1, PB2, and PA. By contrast, two RNAsegments are each transcribed to two mRNAs by splicing. For both the Mand NS genes, coding mRNAs are translated in different reading frames,generating M1 and M2 proteins and NS1 and NS2 proteins, respectively.Increased concentration of free NP triggers the shift from mRNAsynthesis to complementary RNA (cRNA) and viral RNA (vRNA) synthesis.Newly synthesized vRNAs are encapsidated with NP in the nucleus, wherethey function as templates for secondary transcription of viral mRNAs.

Reverse-genetics systems have allowed the manipulation of the influenzaviral genome (Palese, et al. (1996) Proc. Natl. Acad. Sci. USA93:11354-58; Neumann & Kawaoka (1999) Adv. Virus Res. 53:265; Neumann,et al. (1999) Proc. Natl. Acad. Sci. USA 96:9345; Fodor, et al. (1999)J. Virol. 73:9679). Reverse genetics in the influenza virus context is amechanism by which negative sense RNA is engineered into cDNA forrecombinant preparation of organisms having negative strand RNA genomes.The reverse genetics technique involves the preparation of syntheticrecombinant viral RNAs that contain the non-coding regions of thenegative strand virus essential for the recognition of viral RNA byviral polymerases and for packaging signals necessary to generate amature virion. The recombinant RNAs are synthesized from a recombinantDNA template and reconstituted in vitro with purified viral polymerasecomplex to form recombinant ribonucleoproteins (RNPs) which can be usedto transfect cells. See U.S. Pat. Nos. 6,022,726 and 6,001,634,incorporated herein by reference in their entireties.

These recombinant methods allow for the production of influenza virustypes with specific alterations to the polypeptide amino acid sequence.For example, an HA molecule containing a desired substitution may bepart of a recombinant influenza virus. In one method, the recombinantinfluenza virus is made through a genetic engineering method such as the“plasmid only” system (Hoffmann, et al. (2002) Vaccine 20:3165,).

In another method for generating a recombinant virus, an eight plasmidsystem is used, wherein the negative sense RNAs are expressed from a PolI promoter and the coexpression of the polymerase complex proteinsresult in the formation of infectious influenza A virus (Hoffmann, etal. (2000) Proc. Natl. Acad. Sci. USA 97:6108-13). This technologyallows the rapid production of chimeric vaccines from cDNA for use inthe event of an influenza pandemic, and provides the capability toattenuate pathogenic strains (Subbarao, et al. (2003) Virology305:192-200), while eliminating the need to screen reassortant virusesfor the 6:2 configuration (i.e., 6 internal genes and 2 HA and NA genes(one of each gene)). See also U.S. Pat. No. 7,037,707.

In some embodiments, the reassortant viruses are prepared using themethod of Palese et al. ((1996) Proc. Natl. Acad. Sci. USA 93:11354-58),which describes the use of a helper virus system to generate geneticallyengineered virus. In one embodiment, the virus is generated using aninfluenza helper virus method. For example, to construct a 6:2reassortant, an attenuated VN1203 virus could be used as a helper virusto introduce the HA and NA from a second strain. Selection of thetransfectant virus is carried out using neutralizing antibodies againstthe helper HA or NA proteins.

With knowledge of internal genes and HA and NA genes from influenzavirus strains, it will be appreciated that, in an alternativeembodiment, polynucleotides encoding these genes are synthesized bytechniques well know and routinely practiced in the art.

In another embodiment, influenza virus of other influenza A subtypes andinfluenza B viruses are useful in the methods and compositions of theinvention. For example, influenza A virus having any HA subtype iscontemplated, including any of the H1 to H18 subtypes. In still afurther embodiment it is contemplated that an influenza virus having anyof NA subtypes N1 to N11 is useful for the invention.

In certain embodiments, it is contemplated that when generating areassortant, the HA and NA subtype are derived from the same strain, andthe backbone is derived from an influenza virus of the same subtype. Forexample, it is contemplated that any of the following influenza Asubtypes are useful in the invention: H1N1, H2N1, H3N1, H4N1, H5N1,H6N1, H7N1, H8N1, H9N1, H10N1, H11N1, H12N1, H13N1, H14N1, H15N1, H16N1;H1N2, H2N2, H3N2, H4N2, H5N2, H6N2, H7N2, H8N2, H9N2, H10N2, H11N2,H12N2, H13N2, H14N2, H15N2, H16N2; H1N3, H2N3, H3N3, H4N3, H5N3, H6N3,H7N3, H8N3, H9N3, H10N3, H11N3, H12N3, H13N3, H14N3, H15N3, H16N3; H1N4,H2N4, H3N4, H4N4, H5N4, H6N4, H7N4, H8N4, H9N4, H1ON4, H11N4, H12N4,H13N4, H14N4, H15N4, H16N4; H1N5, H2N5, H3N5, H4N5, H5N5, H6N5, H7N5,H8N5, H9N5, H10N5, H11N5, H12N5, H13N5, H14N5, H15N5, H16N5; H1N6, H2N6,H3N6, H4N6, H5N6, H6N6, H7N6, H8N6, H9N6, H10N6, H11N6, H12N6, H13N6,H14N6, H15N6, H16N6; H1N7, H2N7, H3N7, H4N7, H5N7, H6N7, H7N7, H8N7,H9N7, H10N7, H11N7, H12N7, H13N7, H14N7, H15N7, H16N7; H1N8, H2N8, H3N8,H4N8, H5N8, H6N8, H7N8, H8N8, H9N8, H10N8, H11N8, H12N8, H13N8, H14N8,H15N8, H16N8; H1N9, H2N9, H3N9, H4N9, H5N9, H6N9, H7N9, H8N9, H9N9,H10N9, H11N9, H12N9, H13N9, H14N9, H15N9, and H16N9. Influenza A virusesof the following subtypes have been identified previously, H1N1, H2N2,H1N2, H3N2, H3N8, H4N6,H5N1, H5N2, H5N3, H5N9, H6N1, H6N2, H6N5, H7N1,H7N7, H8N4, H9N2, H10N7, H11N6, H12N5, H13N6, H14N5, H15N8, H15N9,H16N3.

The instant modified HA nucleic acids and proteins are of use in anyvirus previously disclosed in the art, e.g., any viruses (Influenza Aand influenza B) previously disclosed or produced, e.g., using backbonessuch as A/Puerto Rico/8/34 (H1N1), A/Ann Arbor/6/60 (H2N2) and B/AnnArbor/1/66, having internal genes from one strain and the HA and NAgenes from a different strain, and in any prior publications referencedherein, including but not limited to: U.S. Pat. Nos. 4,552,758,7,037,707, 7,601,356, 7,566,458, 7,527,800, 7,510,719, 7,504,109,7,465,456, 7,459,162; US 2009/0297554, US 2009/0246225, US 20090208527,US 2009/0175909, US 2009/0175908, US 2009/0175907, US 2009/0136530, US2008/0069821, US 2008/0057081, US 2006/0252132, US 2006/0153872, US2006/0110406, US 2005/0158342, US 2005/0042229, US 2007/0172929, WO2017/070620, WO 2008/157583, WO 2008/021959, WO 2007/048089, WO2006/098901, WO 2006/063053, WO 2006/041819, WO 2005/116260, WO2005/116258, WO 2005/115448, WO 2005/062820, WO 2003/091401 and anyviruses identified therein as useful for the FLUMIST™ vaccine, which maycontain internal genes from one influenza A subtype and HA and NA genesof the same subtype in a recombinant virus. All such documents areincorporated by reference herein in their entirety.

Cells Lines. Typical influenza viruses are adapted for growth in chickeneggs but the expense for maintaining the egg cultures is significantlygreater than growing virus in cell culture. Conventional chicken embryocell (CEC) cultures have been used in attempts to grow influenza virusfor vaccine, but these provide only some of the protease activities of awhole chicken embryo and, hence, allow replication of a limited range ofinfluenza virus strains. Standard procedures for preparation of CECcultures involve removal of the head and inner organs and multipletrypsinization steps. These procedures result specifically in the lossof brain, heart, lung, liver and kidney cells, which have been shown toreplicate a number of influenza strains (Scholtissek, et al. (1988) J.Gen. Virol. 69:2155-2164). Standard procedures thus result in a highlyselected cell population consisting mainly of fibroblasts, which arelimited in terms of the virus strains that they can support.

Improvements in influenza virus production have been achieved in bothchicken cultures and in mammalian cell lines. For instance, it has beenreported that the limited replication of several influenza A strains instandard cell cultures could be ameliorated by the addition of trypsinto the tissue culture medium. For example, trypsin additionsignificantly increases the infectivity of various strains grown in CECcultures (Lazarowitz, et al. (1975) Virology 68:440-454). In addition,Stieneke-Grober, et al. ((1992) EMBO J. 11:2407-2414) have identifiedthe HA activating enzyme in MDBK cells as a furin-like protease. Suchenzymes have been isolated from human and mouse tissues and constitute anew family of eukaryotic subtilisin-like endoproteases. Vero cellsadapted for improved viral growth and vaccine production are describedin U.S. Pat. No. 6,146,873 and Kistner, et al. ((1998) Vaccine16:960-8). Additional mammalian cell lines useful for culture and growthof virus for use in vaccines include, but are not limited to, MRC-5,MRC-9, Lederle 130, Chang liver and WI-38 (human fibroblast); U937(human monocyte); Vero and CV-1 (African Green monkey): IMR-90 andIMR-91 (human lung fibroblast having characteristics of smooth muscle),MUCK (Madin Darby canine kidney), MDBK (Madin Darby bovine kidney), HEK293T (human embryonic kidney), H9, CEM and CD4-expressing HUT78 (human Tcell): PerC6 (human retinoblast); BHK-21 cells (baby hamster kidney),BSC (monkey kidney cell); and LLC-MK2 (monkey kidney). In accordancewith the present invention, an HA2 protein encoded by a nucleic acidmolecule modified to include an A->T mutation in the first position ofthe codon for Thr436 thereby eliminating m⁶A at this position provides asignificant increase in HA RNA expression in mammalian cells. Therefore,in particular aspects, a host cell of the invention is a mammalian hostcell.

Vaccines. It is contemplated that a desired virus strain obtained fromcell-culture preparation is used to produce a vaccine. Many types ofviral vaccines are known, including but not limited to, attenuated,inactivated, subunit, and split vaccines.

Attenuated vaccines are live viral vaccines that have been altered insome manner to reduce pathogenicity and no longer cause disease.Attenuated viruses are produced in several ways, including growth intissue culture for repeated generations and genetic manipulation tomutate or remove genes involved in pathogenicity. For example, in oneembodiment, viral genes and/or proteins identified as involved inpathogenicity or involved in the disease manifestation, are mutated orchanged such that the virus is still able to infect and replicate withina cell, but it cannot cause disease. An example of this is to mutagenizethe HA1/HA2 cleavage site. Attenuation of virus has also been successfulby insertion of a foreign epitope into a viral gene segment, for examplethe NA gene (Castrucci, et al. (1992) J. Virol. 66:4647-4653), therebyinterfering with the normal function of the genome. Viruses are alsoattenuated using cold adaptation methods well-known in the art. See, forexample, Maassab, et al. ((1999) Rev. Med. Virol. 9:237-44), whichdiscusses methods to attenuate type A and Type B influenza virus, andGhendon, et al. ((2005) Vaccine 23:4678-84), which describe a coldadapted influenza virus that grows in MDCK cells.

Additional methods to attenuate a virus include construction of areassortant virus lacking the NS1 gene. See for example U.S. Pat. Nos.6,468,544, 6,573,079, 6,669,943, 6,866,853, US 2003/0157131 and US2004/0109877, which disclose an attenuated virus lacking a functionalNS1 gene. The NS1 gene may be completely deleted or partially deleted oraltered by mutation such that there is no functional expression of theNS1 gene in the virus. Virus particles lacking the NS1 gene demonstratean attenuated phenotype compared to wild-type virus.

After production of the attenuated virus, the vaccine is prepared usingstandard methods. The virus is purified using standard methods known inthe art, for example using size exclusion chromatography, high-speed(ultra) centrifugation or sucrose gradients.

Subunit vaccines are killed vaccines. Production of subunit vaccineinvolves isolating a portion of the virus that activates the immunesystem. In the case of influenza, subunit vaccines have been preparedusing purified HA and NA, but any mixture of viral proteins is used toproduce a subunit vaccine. Generally, the viral protein, such as HA isextracted from recombinant virus forms and the subunit vaccine isformulated to contain a mixture of these viral proteins from differentstrains.

Split vaccines are killed vaccines produced by treating an envelopedvirus with detergent to solubilize the proteins in the envelope. In thecase of influenza virus, HA and NA become solubilized. In oneembodiment, nonionic detergents are used for producing split vaccines.Examples of non-ionic detergents, include, but are not limited to,Nonanoyl-N-Methylfucamide (Mega 9), Triton™ X-100, Octylglucoside,Digitonin, C12E8, Lubrol, NONIDET® P-40, and polysorbate (for examplepolysorbate 20, 80 or 120).

Inactivated viral vaccines are prepared by inactivating the harvestedvirus and formulating it using known methods for use as a vaccine toinduce an immune response in a mammal. Inactivation is carried out usingagents including but not limited to formaldehyde, UV irradiation,glutaraldehyde, binary ethyleneimine (BEI), and beta-propiolactone.Inactivating agents are used at a concentration high enough toinactivate substantially all viral particle in the solution. By way ofexample and without limitation, virus inactivation with gammairradiation is described in U.S. Pat. No. 6,254,873; inactivation withformalin is described in U.S. Pat. Nos. 6,254,873 and 6,635,246;inactivated with formaldehyde has been described for JE-VAX®, Japaneseencephalitis virus vaccine (Couch, et al. (1997) J. Infect. Dis.176(Suppl 1):538-44); photodynamic inactivation by visible light(Wallis, et al. (1963) J. Immunol. 91:677-682); inactivation with UVlight (WO/2008/039494); chlorine inactivation; inactivation withwater-insoluble, hydrophobic polycations, e.g., N,N-dodecylmethyl-polyethylenimine (PET) (Halder, et al. (2006) Proc. Natl. Acad.Sci. USA 103:17667-17671); thermal inactivation (Thomas, et al. (2007)J. Food Protect. 70:674-680); inactivation with betapropiolactone isdescribed in European Pharmacopoeia 5.0; and inactivation by binaryethylenimine is described in U.S. Pat. No. 6,803,041. It is contemplatedthat during the inactivation step, purification of subunits, and/orsplitting is performed before or after purification of the virus fromcell culture. For example, production of an inactivated virus vaccine,may involve removal of cellular material, inactivation of virus,purification and solubilization of the viral envelope. In oneembodiment, a reassortant virus described herein is grown and isolatedfrom Vero cells as described in Kistner, et al. ((2007) Vaccine25:6028-36).

In some embodiments, the nucleic acid molecule (e.g., mRNA) of thisinvention may be used as the vaccine, wherein the vaccine is optionallyformulated in a lipid nanoparticle (e.g., a lipid nanoparticle composedof a cationic lipid, a PEG-modified lipid, a sterol and a non-cationiclipid). See, e.g., WO 2017/070620 or WO 2017/070622.

A vaccine is then prepared using standard adjuvants and vaccinepreparations known in the art. Adjuvants include, but are not limitedto, saponin, non-ionic detergents, vegetable oil, mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions,keyhole limpet hemocyanins, and potentially useful human adjuvants suchas N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-s-n-glycero-3-hydroxyphosphoryloxy)-ethylamine,BCG (bacille Calmette-Guerin) Corynebacterium parvum, ISCOMs,nano-beads, squalene, and block copolymers, which are contemplated foruse alone or in combination. ISCOMs or Immune Stimulating Complexes area novel vaccine delivery system and are unlike conventional adjuvants(Morein, et al. (1984) Nature 308:457-460). An ISCOM is formed in twoways: (1) the antigen is physically incorporated in the structure duringits formulation; or (2) an ISCOM-matrix does not contain antigen but ismixed with the antigen of choice by the end-user prior to immunization.After mixing, the antigens are present in solution with the ISCOM-matrixbut are not physically incorporated into the structure.

In one embodiment, the adjuvant is an oil-in-water emulsion.Oil-in-water emulsions are well known in the art and have been suggestedto be useful as adjuvant compositions (EP 399843; WO 95/17210, US2008/0014217). Ideally, the oil is present in an amount of 0.5% to 20%(final concentration) of the total volume of the antigenic compositionor isolated virus, at an amount of 1.0% to 10% of the total volume, orin an amount of 2.0% to 6.0% of the total volume. In some embodiments,oil-in-water emulsion systems useful as adjuvant have a small oildroplet size. For example, the droplet sizes will be in the range 120 to750 nm, or from 120 to 600 nm in diameter.

In order for any oil-in-water composition to be suitable for humanadministration, the oil phase of the emulsion system includes ametabolizable oil. The oil may be any vegetable oil, fish oil, animaloil or synthetic oil, which is not toxic to the recipient and is capableof being transformed by metabolism. Nuts, seeds, and grains are commonsources of vegetable oils. Synthetic oils are also part of thisinvention and can include commercially available oils such as NEOBEE®and others. A particularly suitable metabolizable oil is squalene.Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene)is an unsaturated oil which is found in large quantities in shark-liveroil, and in lower quantities in olive oil, wheat germ oil, rice branoil, and yeast, and is a particularly suitable oil for use in thisinvention. Squalene is a metabolizable oil by virtue of the fact that itis an intermediate in the biosynthesis of cholesterol (Merck index, 10thEdition, entry no. 8619). Exemplary oils useful for an oil in wateremulsion, include, but are not limited to, sterols, tocols, andalpha-tocopherol.

In additional embodiments, immune system stimulants are added to thevaccine and/or pharmaceutical composition. Immune stimulants include:cytokines, growth factors, chemokines, supernatants from cell culturesof lymphocytes, monocytes, or cells from lymphoid organs, cellpreparations and/or extracts from plants, cell preparation and, orextracts from bacteria (e.g., BCG, mycobacterium, Corynebacterium),parasites, or mitogens, and novel nucleic acids derived from otherviruses, or other sources (e.g. double stranded RNA, CpG) blockco-polymers, nano-beads, or other compounds known in the art, used aloneor in combination.

Particular examples of adjuvants and other immune stimulants include,but are not limited to, lysolecithin; glycosides (e.g., saponin andsaponin derivatives such as QUIL-A® (QS7 and QS21) or GPI-0100);cationic surfactants (e.g., DDA); quaternary hydrocarbon ammoniumhalogenides; pluronic polyols; polyanions and polyatomic ions;polyacrylic acids, non-ionic block polymers (e.g., PLURONIC® F-127); and3D-MPL (3 de-O-acylated monophosphoryl lipid A). See, e.g., US2008/0187546 and US 2008/0014217.

Pharmaceutical Formulations and Administration. The administration ofthe vaccine composition is generally for prophylactic purposes. Theprophylactic administration of the composition serves to prevent orattenuate any subsequent infection. A “pharmacologically acceptable”composition is one tolerated by a recipient patient. It is contemplatedthat an effective amount of the vaccine is administered. An “effectiveamount” is an amount sufficient to achieve a desired biological effectsuch as to induce enough humoral or cellular immunity. This may bedependent upon the type of vaccine, the age, sex, health, and weight ofthe recipient. Examples of desired biological effects include, but arenot limited to, production of no symptoms, reduction in symptoms,reduction in virus titer in tissues or nasal secretions, completeprotection against infection by influenza virus, and partial protectionagainst infection by influenza virus.

A vaccine or composition of the present invention is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient patient that enhances at least one primary orsecondary humoral or cellular immune response against at least onestrain of an infectious influenza virus. The vaccine composition isadministered to protect against viral infection. The “protection” neednot be absolute, i.e., the influenza infection need not be totallyprevented or eradicated, if there is a statistically significantimprovement compared with a control population or set of patients.Protection may be limited to reducing the severity or rapidity of onsetof symptoms of the influenza virus infection.

In one embodiment, an attenuated or inactivated vaccine composition ofthe present invention is provided either before the onset of infection(so as to prevent or attenuate an anticipated infection) or after theinitiation of an actual infection, and thereby protects against viralinfection.

In one aspect, methods of the invention include a step of administrationof a pharmaceutical composition. The virus, antigenic composition orvaccine is administered in any means known in the art, including viainhalation, intranasally, orally, and parenterally. Examples of parentalroutes of administration include intradermal, intramuscular,intravenous, intraperitoneal and subcutaneous administration.

Ideally, influenza vaccine administration is based on the number ofhemagglutinin units (HAU) per dose. One HAU is defined as the quantityof antigen required to achieve 50% agglutination in a standardhemagglutinin assay with chicken red blood cells. Avian influenza virusvaccines as described herein are effective in formulations comprising HAunits (HAU) between about 10 ng and about 1 μg, between about 20 ng andabout 500 ng, between about 50 ng and about 250 ng, between about 75 ngand about 200 ng, about 100 ng, about 125 ng, about 150 ng, or about 175ng. In a related embodiment, a vaccine composition composed of aninactivated virus includes an amount of virus corresponding to about 0.1to about 200 μg of hemagglutinin protein/ml, or any range or valuetherein. In a related embodiment, a vaccine composition of the presentinvention includes from about 10² to 10⁹ plaque forming units (PFU)/ml,or any range or value therein, where the virus is attenuated. In someembodiments, the vaccine composition includes about 10², about 10³,about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸ or about 10⁹PFU/ml. It is further contemplated that the vaccine composition includesfrom 10² to about 10⁴ PFU/ml, from about 10⁴ to about 10⁶ PFU/ml, orfrom about 10⁶ to about 10⁹ PFU/ml.

In another aspect. inactivated flu vaccine is quantified by a singleradial diffusion (SRD) assays (see Kistner, et al. (2007) Vaccine25:6028-36; Wood, et al. (1997) J. Biol. Stand. 5:237-247) and expressedin micrograms hemagglutinin (per ml or per dose). In one embodiment, thedose of a seasonal vaccine is 15 μg per strain, 45 μg in total in threedosages. For (pre)pandemic vaccines the dose typically depends on theadjuvant. In one aspect, the dose range is 1 μg to 15 μg per vaccine,and in some preparations, up to 75 μg per vaccine are useful. In oneembodiment, the vaccine dose is administered at a dose from 1 μg to 100μg HA. In a further embodiment, the vaccine comprises an HA content of 1μg to 30 μg per vaccine. In related embodiments, the vaccine dose isadministered at 1 μg, at 3 μg, at 5 μg, at 7.5 μg, at 10 μg, at 12.5 μg,at 15 μg, at 20 μg, at 25 μg, or at 30 μg HA, or in any amount up to 100μg as necessary. It is contemplated that, in some embodiments, the doseof vaccine is adjusted based on the adjuvant used for vaccinepreparation.

Accordingly, single vaccine dosages include those having about 1 μg,about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg,about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, about 13μg, about 14 μg, about 15 μg, about 16 μg, about 17 μg, about 18 μg,about 19 μg, about 20 μg, about 21 μg, about 22 μg, about 23 μg, about24 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg,about 50 μg, about 55 μg, about 60 μg, about 65 μg, about 70 μg, about75 μg, about 80 μg, about 85 μg, about 90 μg, about 95 μg, about 100 μg,and more than 100 μg hemagglutinin provided in single or multipledosages at the same or different amount of hemagglutinin.

It is further contemplated that in certain embodiments, the virus orantigenic composition is administered in doses including similar HAunits or pfu as contemplated for vaccine administration.

When administered as a solution, the vaccine is prepared in the form ofan aqueous solution. Such formulations are known the art and areprepared by dissolution of the antigen and other appropriate additivesin the appropriate solvent. Such solvents include water, saline,ethanol, ethylene glycol, and glycerol, for example. Suitable additivesinclude certified dyes and antimicrobial preservatives, such asthimerosal (sodium ethylmercuithiosalicylate). Such solutions may bestabilized using standard methods, for example, by addition of partiallyhydrolyzed gelatin, sorbitol, or cell culture medium and may be bufferedusing standard methods, using, for example reagents such as sodiumhydrogen phosphate, sodium dihydrogen, phosphate, potassium hydrogenphosphate and/or potassium dihydrogen phosphate or Tris. Liquidformulations may also include suspensions and emulsions. The preparationof suspensions includes, for example, using a colloid mill, andemulsions include, for example, using a homogenizer.

The following non-limiting examples are provided to further illustratethe present invention.

Example 1: Identification and Mutation of Conserved m6A Sites in HA mRNASequences

Nucleic acid sequences encoding HA from multiple influenza virus strainswere analyzed for m6A sites using a computational predictor of mammalianm6A site named SRAMP (Zhou, et al. (2016) Nucleic Acids Res.44(10):e91). The aligned HA sequences were from viruses isolated fromavian, swine, and human over the past 80 years. This analysis identifiedthree clusters of m⁶A sites with cluster 1 located at nucleotidepositions 764, 774, 785 and 795; cluster 2 located at nucleotidepositions 1331 and 1338; and cluster 3 located at nucleotide positions1380 and 1395. To determine the effects of m⁶A modifications at theseclusters on viral replication and infectivity, nucleotide site mutationswere performed to remove the m⁶A on the HA mRNA sequence. In particular,nucleotides 764, 774, 785, 795 (mutant 1); nucleotides 1331, 1338(mutant 2); and nucleotides 1380, 1395 (mutant 3) of clusters 1, 2 and 3were respectively mutated to remove the sites for m⁶A modifications. Inlight of the mutations at nucleotides 774, 1338 and 1380, the resultingHA proteins included threonine to serine mutations at the correspondingpositions in the HA protein sequence.

Reverse genetics was used to generate viruses using the mutant 2 HA genesequence. In particular, seven influenza gene plasmids corresponding toA/Puerto Rico/8/1934 (Matrix, PB1, PB2, PA, NS, NP, NA) were transfectedinto a co-culture of MDCK and 293T cells along with a mutant 2 HAplasmid. Supernatant was harvested from transfected cells 48 hours laterand injected into embryonic chicken eggs to generate mutant virus stock.In vitro single (4 to 8 hours at multiplicity of infection (MOI) 10) andmultiple replication cycles (12 to 48 hours at MOI 0.1) were conductedusing a human lung epithelial cell line (A549). Nuclear and cytoplasmicRNA was extracted from A549 cells; vRNA and mRNA HA, matrix and PB1 geneexpression levels were determined by RT-PCR; and RNA levels werenormalized to wild-type levels at 4 hours (single replication cycle) or12 hours (multiple replication cycles). In addition, supernatant washarvested at 8 hours from A549 cells infected at MOI 10 and infectiousviral titers were measured by plaque assay.

The results of this analysis indicated that the cluster 2 mutant (mutant2) showed a significant increase in HA RNA expression over the course ofa single replication round (FIG. 1 ). Notably, mutant 2 RNA wassynthesized in the nucleus and successfully trafficked to the cytoplasm.As with HA RNA expression, the cluster 2 mutant (mutant 2) showed asignificant increase in matrix and PB1 RNA expression over the course ofa single replication round, thereby demonstrating that the mutations inthe HA gene not only affected HA gene expression but also othernon-mutated influenza genes. Consistent with an increase in RNAexpression, the cluster 2 mutant (mutant 2) showed a significantincrease in HA and matrix protein levels over the course of a singlereplication round. Further, cluster 2 mutant (mutant 2) showed asignificant increase in infectious viral titers over the course of asingle replication round (FIG. 2 ) thereby demonstrating that increasedgene expression of cluster 2 mutant also resulted in an increase ininfectious viral titer over a single replication cycle.

Analysis of HA and matrix expression levels after multiple replicationcycles indicated that cluster 2 mutant (mutant 2) showed a significantincrease in RNA expression over the course of multiple replicationrounds thereby demonstrating that increased gene expression wassustained over time (FIG. 3 ). Consistent with an increase in RNAexpression, the cluster 2 mutant (mutant 2) showed a significantincrease in HA and matrix protein levels over the course of multiplereplication rounds as well as a significant increase in infectious viraltiters (FIG. 4 ).

To further characterize the mutant 2 virus, 7-week-old Balb/CJ mice wereinfected with 10⁴ median tissue culture infectious dose (TCID₅₀) or 10³TCID₅₀ of wild-type or mutant 2 virus. Mice were weighed daily for 14days and euthanized at human endpoints. The results of this analysisindicated that compared to wild-type, which had a lethal dose 50 (LD₅₀)of 1.10E+04, the mutant 2 virus exhibited increased lethality (LD₅₀ of1.40E+03) at the same dose as compared to wild-type.

Viral lung titers were also analyzed by infecting 7-week-old Balb/CJmice with 10⁴ TCID₅₀ of virus and harvesting lung tissue at days 2, 4,and 6 days post-infection. The lung tissue was collected in 500 μL ofphosphate buffered saline (PBS), homogenized, and centrifuged a 500×gfor 20 minutes. The supernatant was collected and viral titers in thelysates were determined by TCID₅₀ on MDCK monolayers. This analysisindicated that the mutant 2 viral titers peaked earlier at 2 dayspost-infection compared to 4 days post-infection for the wild-type virusand mutant 2 viral titers peaked at a higher level compared to wild-typevirus (FIG. 5 , Panel A). Lung tissue was also dissected to assessactive infection area. Lungs were inflated with 1 mL of 10%neutral-buffered formalin and then placed in 3 mL of formalin for 72hours to complete fixation. Lungs were embedded in paraffin blocks andsectioned onto glass slides, and serial tissue sections were stainedwith haematoxylin and eosin for histology. Histologic grading of lesionswas completed by a pathologist blinded to treatment group. Pulmonarylesions were assigned scores based on the basis of their severity andextent as follows: 0, no lesions; 1, minimal, focal to multifocal,barely detectable; 15, mild, multifocal, small but conspicuous; 40,moderate, multifocal, prominent; 80, marked, multifocal coalescing,lobar; 100, severe, diffuse, with extensive disruption of normalarchitecture and function. The results of this analysis indicated thatmutant 2 virus spread more extensively in the lung (FIG. 5 , panel B),which was consistent with increased mortality.

For immunological characterization of the mutant 2 virus, A549 cellswere infected at an MOI of 3; cells were harvested at 3, 6, 9, 12, 24,and 48 hours post-infection; and RNA was extracted using a Qiagen RNeasyKit. cDNA was prepared for quantitative PCR (qPCR) using the FluidigmBiomark system. Data were collected using the Fluidigm Biomark datacollection software and normalized to wild-type levels at 3 hourspost-infection. Two-way ANOVA was performed for statistical analysis.This analysis indicated that levels of key type I interferons (ISG15,IFIT1) were significantly increased in cells infected with mutant 2virus compared to wild-type virus. Increases in ISG15 and IFIT1expression occurred at the onset of infection and were sustained for atleast a minimum of 24 hours after infection. In addition, Type IIIinterferon (IFNL2) and key inflammatory cytokine (TNFA) and chemokine(CXCL10) RNA levels were significantly increased in cells infected withmutant 2 virus compared to wild-type virus. Overall, mutant 2 virusinduced a stronger immunological response compared to wild-type in humanepithelial cells.

Further immunological characterization of the mutant 2 virus was carriedout in viva. Mice were infected with 10⁴ TCID50 mutant 2 virus orwild-type. At 2 days post-infection, mouse lungs were harvested andflash-frozen in liquid nitrogen. Lungs were then homogenized in 500 μLof PBS and lysate was collected after centrifugation at 500×g for 20minutes. Cytokine and chemokine levels were measured using MilliplexMouse Cytokine/Chemokine Magnetic Premixed 32 plex Bead Panelimmunoassay per manufacture's protocol. The results of this analysisindicated that the mutant 2 virus induced a significant increase in theproduction of cytokines and chemokines at 2 days post-infection comparedto wild-type virus (FIG. 6 ). Therefore, mutant 2 virus infection leadsto an increased immunological response in mice early in infectioncompared to wild-type virus.

1. A modified nucleic acid molecule encoding influenza virushemagglutinin, wherein said nucleic acid molecule comprises an A->Tmutation in the first position of codon 436 of the hemagglutinin andoptionally a C->T mutation in the third position of codon 433 of thehemagglutinin.
 2. The modified nucleic acid molecule of claim 1, whereinsaid nucleic acid molecule encodes a modified hemagglutinin proteinhaving a Thr436Ser amino acid substitution.
 3. A vector comprising themodified nucleic acid molecule of claim
 1. 4. A host cell comprising themodified nucleic acid molecule of claim
 1. 5. A modified influenza virushemagglutinin protein comprising a Thr436Ser amino acid substitution. 6.A vaccine comprising the modified nucleic acid molecule of claim
 1. 7.The vaccine of claim 6, further comprising an adjuvant.
 8. A vaccinecomprising the modified influenza virus hemagglutinin protein of claim5.
 9. The vaccine of claim 8, further comprising an adjuvant.
 10. Amethod for the production of influenza virus, comprising (a)transfecting host cells with a modified nucleic acid molecule comprising(i) a C->T mutation in the third position of codon 433 of thehemagglutinin; (ii) an A->T mutation in the first position of codon 436of the hemagglutinin; or (iii) a combination of (1) and (11), (b)incubating the host cells under conditions that allow for influenzavirus to be produced, and (c) isolating the influenza virus from thehost cells.
 11. The method of claim 10, wherein the host cells aremammalian cells.
 12. The method of claim 11, wherein the mammalian cellsare a cell line selected from the group of MRC-5, MRC-9, Lederle 130,Chang liver, WI-38, U937, Vero, CV-1, IMR-90, IMR-91, MDCK, MDBK, HEK293T, H9, CEM, CD4-expressing HUT78, PerC6, BHK-21, BSC, and LLC-MK2.